1. Field of the Invention
This invention relates to novel sparingly soluble salts and immobilized ionic complexes of the meriquinone oxidation products of benzidine and substituted benzidines and to their use as analytical visualization signals in a wide array of chemical, biological and clinical tests.
2. Description of Background and Related Art
Peroxidative oxidation usually occurs according to one or the other of the following reaction schemes: AH.sub.2 +ROOH.fwdarw.A+ROH+H.sub.2 O; H.sub.2 O+AH.sub.2 +ROOH.fwdarw.AH.sub.2.sup.+2 +ROH+2OH.sup.- ; in which AH.sub.2 is a hydrogen donor and ROOH is a hydroperoxide. (Which reaction is preferred depends on the base strength of A and on the reaction pH.) Over the years, many analyses for peroxidative activity have been developed. Some are intended to identify, locate or quantitate peroxides either as compounds of interest or, in the case of hydrogen peroxide, as the product of oxidation by molecular oxygen, especially those catalyzed by a class of enzymes known as oxidases. Other such methods are intended to identify, locate, or quantitate catalysts of peroxidative activity, such as transition-metal ions, hemes, hemoproteins, and the peroxidase enzymes. Among the latter, two classes of use predominate over all others: (a) the analysis of hemoglobin in forensic specimens, feces, urine, and cell-free blood plasma or serum, and (b) the analysis of peroxidase employed as a label in binding assays.
The first class of assays is not very sensitive, because non-peroxidase hemoproteins and isolated hemes are inefficient peroxidative catalysts. Furthermore, they are subject to interference from contaminating transition-metal ions, principally iron and copper, and occasionally from contaminating peroxidase enzymes. Nevertheless, they are so simple and their diagnostic relevance is so great that they are popular in their respective fields of use. For example, many commercial clinical tests for occult blood in fecal material exist for screening for cancer and pre-cancerous growths in the colon.
The second class of assays can be extremely sensitive, because horseradish peroxidase not only is much more efficient catalytically than other hemoproteins, but also is one of the most efficient enzymes capable of producing colored products suitable for spectrophotometric or fluorometric analysis. They also can be highly specific, because peroxidase enzymes occur naturally in relatively few clinical or biological samples, and the potential transition-metal-ion interference usually can be blocked with chelating agents.
A key variable in the design of peroxidase-linked assays is the choice of chromogenic substrate, for several reasons:
(a) Enzyme catalytic efficiency ranges over several orders of magnitude, depending on the structure of the hydrogen donor.
(b) Some spectral changes accompanying oxidation are more sensitive than others, having larger extinction coefficient changes or occuring in more easily detected spectral regions.
(c) Some colored products are soluble; others are insoluble. The former are desirable for instrumental analyses of product absorbance or fluorescence in solution. The latter are essential for assays in which the signal should be localized or trapped in a gel or on a solid surface.
(d) Many peroxidase substrates, phenols and aromatic amines, are known or thought to be mutagenic or carcinogenic.
Benzidine and several substituted benzidine derivatives were developed as peroxidase substrates consumed with higher turnover numbers than many other aromatic amines and phenols, giving convenient and large absorbance changes in the visible spectral region. Most can give water-insoluble products, usually polymeric in nature. However, most are known or thought to be carcinogenic or mutagenic. 3,3',5,5'-tetramethylbenzidine (TMB) was developed as a non-carcinogenic peroxidase substrate (e.g., Holland et al. (1974) Tetrahedron, 30: 3299-3302). For this reason, and because it also appears to be one of the most sensitive peroxidase substrates, it has rapidly found widespread use (a) in enzyme immunoassays in which the product color is measured spectrophotometrically in solution (e.g., Bos et al. (1981) Journal of Immunoassay, 2: 187-204), (b) in solution-phase spectrophotometric determination of hemoglobin (e.g., Liem et al. (1979) Analytical Biochemistry, 98: 388-395), (c) in solid-phase spectrophotometric determination of hemoglobin (e.g., Burkhardt et al. (1981) European Patent Application No. 81104634.1; U.S. Pat. No. 4,447,542) or drugs (via peroxidase-linked specific binding assay, U.S. Pat. No. 4,447,529), (d) in detection of hemoglobin, other hemoproteins, or oxidases in electrophoretic gels (e.g., Thomas et al. ( 1976) Analytical Biochemistry, 75: 168-176), and (e) in neurohistochemistry (e.g., Mesulam (1978) Journal of Histochemistry and Cytochemistry, 26: 106-117).
Conspicuously scarce in the documentary record are reports of TMB as a peroxidase substrate in common enzyme-linked solid-phase assays such as immunohistochemical staining, Western blots, Southern blots, or immunodot blots, where less sensitive and more hazardous HRP substrates forming insoluble products have been used (e.g., Hawkes et al. (1982) Analytical Biochemistry, 119: 142-147). Although there are no published applications of TMB to Southern, Northern, Western, or immunodot blots, Trojanowski et al. (1983) Journal of Histochemistry and Cytochemistry, 31: 1217-1223 reported its immunohistochemical use in a comparison with several other chromogens which clearly form insoluble products: diaminobenzidine, aminoethylcarbazole, o-tolidine, and a mixture of paraphenylene diamine and pyrocatechol. However, TMB was judged to be one of the least effective substrates, being less sensitive than diaminobenzidine (despite the opposite finding in neurohistochemical studies) and giving crystals sufficiently large to obscure microstructural detail.
In several of the other solid-phase or gel-phase studies cited above, the TMB signal was also not completely satisfactory. Fujii et al. (1984) Neuroscience Research, 1: 153-156 cited the instability of the TMB product in the absence of special fixatives, and Olsson et al. (1983) J. Neuroscience Methods, 7: 49-59 and many other neurohistochemists mentioned the tendency of the TMB product to form crystals so large that they obscured fine detail. In an histochemical effort to observe the localization of native peroxidase in cross-sections of plant stems, the TMB-generated color was observed to be unstable (Imberty et al. (1984) Plant Science Letters, 35: 103-108). Broyles et al. (1979) Analytical Biochemistry, 94: 211-219 found the TMB product to be mobile in the stained electrophoretic gel, frustrating either quantitation or maintenance of a permanent record. Both Broyles et al. and Francis et al. (1984) Analytical Biochemistry, 136: 509-514 noted a colored background in TMB-stained gels which must represent peroxidative catalysis by impurities in the gel or the reagents. In the other cited class of solid-phase assays of hemoglobin or peroxidase (e.g., Burkhardt et al., supra), the assay interval was so short that physical form of liability of the TMB product would be unlikely to influence the outcome.
Removal of two electrons from benzidine or a substituted benzidine (e.g., by peroxidative oxidation) creates an oxidized product called a quinone diimine. The blue reaction product of TMB oxidation has been reported to exist largely as a charge-transfer complex between one TMB molecule and one quinone diimine, having an average oxidation state halfway between those of its two components and called a meriquinone (Josephy et al. (1982) J. Biol. Chem., 257: 3668-3675). In that paper, both the meriquinone and the quinone diimine were represented as neutral molecules, as had also been the case in an earlier paper on the mechanism of ortho-dianisidine oxidation (Claiborn and Fridovich (1979) Biochemistry, 18: 2324-2329). Later the quinone diimine and meriquinone formed from benzidine oxidation were drawn as dications (Josephy et al. (1983) J. Biol. Chem., 258: 5561-5569), although no pK.sub.a values for meriquinones or quinone diimines have been reported.
Before the discovery of TMB, Straus (1963) Journal of Histochemistry and Cytochemistry, 12: 462-469 observed that unspecified buffer salts caused the blue product of benzidine oxidation (meriquinone) to form crystals of undetermined composition. Other solid-phase or gel-phase applications of TMB as a peroxidative substrate, cited above, used buffers the anions of which (acetate, citrate and phosphate) applicants have shown to form relatively soluble salts of the TMB meriquinone. While Mesulam, supra, Olsson et al., supra, and other neurohistochemists reported the deposition of the blue product of TMB oxidation as crystals (of undetermined composition) from acetate and phosphate buffers in neurohistochemical applications, most of the cited references on solid-phase and gel-phase applications disclose no clear evidence of product precipitation or immobilization. In fact, the opposite result was reported by Broyles et al., supra. As recently as 1983, Josephy et al. (Journal of Biological Chemistry, 285: 5561-5569) cited the observation of Broyles et al. regarding unsatisfactory solubility properties of the TMB meriquinone. Neurohistochemists have used methyl salicylate (Adams (1980) Neuroscience Letters, 17: 7-9), ammonium molybdate (Fujii et al., supra), potassium ferricyanide (Albers et al. (1984) Journal of Histochemistry and Cytochemistry, 32: 1005-1008), or sodium nitroprusside (e.g., Mesulam, supra) to stabilize the blue TMB product. However, the molecular basis of these effects is unknown. Methyl salicylate is non-ionic in the pH range used, and the other three stabilizers can undergo reduction reactions or serve as sources of anions which might precipitate the meriquinone. U.S. Pat. No. 4,525,452 describes the isolation of unoxidized TMB as a solid sulfate or dichloride salt, but no reference is made to oxidized TMB.
The principal reported difficulties in applying TMB as a peroxidative substrate in solid-phase or gel-phase assays are (a) excessive solubility of the colored reaction product, (b) lack of control of crystallization in the neurological and immunohistochemical staining applications where insoluble product is obtained but large crystals can obscure cellular microstructures, (c) excessive background oxidation of TMB by contaminants, and (d) "fading" of the meriquinone color for unspecified reasons.
Van Duijn Receuil des Travaux Chimiques des Pays-Bas (1955) 74: 771-778 disclosed that inorganic Cl.sup.- and SO.sub.4.sup.-2 salts precipitated the blue meriquinone intermediate of benzidine oxidation. The precipitates were not shown to be ionic nor precipitation shown to be complete. In addition, no quantitation of meriquinone solubility was made. Weis Chemistry and Industry (1938) 16: 517-518 disclosed nonexperimental suggestions that the blue compounds observed by Schlenk and by Barzilowsky were semiquinones, not charge-transfer complex meriquinones, that the semiquinones should be mono-cations, and that the blue solids obtained with various anions should be salts. Schlenk Annalen der Chemie (1908) 363: 313-339 disclosed blue chloride "salts" of the meriquinones of 3,3'-dichloro-5,5'-dimethylbenzidine and 3,3'-dimethylbenzidine precipitated from water. No measurement of solubility or proof of the ionic nature of the solids was obtained. Barzilowsky Chemikes-Zeitung (1905) 29: 292 disclosed the salt of ferrocyanide tetraanion and two meriquinone dications. No measurement of solubility was made.
These four publications and that of Straus, supra, show that although the term, "salt", has been used to describe the blue precipitate formed when certain inorganic or unspecified salts were added in high or unspecified concentration to the blue product of oxidation of benzidine or several substituted benzidines, there has been no demonstration of the generality of the phenomenon, of the quantitative controllability of the phenomenon, of the solubilities of the products, or of the ionic nature of the product. The focus of this early work was on the structures of the blue dyes, but there was not even consensus on their chemical structure.
Although benzidine and substituted benzidines are most commonly used as chromogenic electron donors in oxidation by peroxide, at least some oxidase enzymes catalyze TMB oxidation by molecular oxygen (Miller and Nicholas (1984) Analytical Biochemistry, 140: 577-580). This fact is a reminder that improvements in the technology of visualizing oxidative reactions with benzidine or substituted benzidines have broad applicability beyond the field of peroxidase-based assays.